Method for preparing poultry testicular interstitial cells

A technology for Leydig cells and poultry is applied in the field of preparation of Leydig cells of avian species, which can solve the problems of few cells, low viability and low purity, and achieve the effect of a good cell working platform.

Active Publication Date: 2018-06-22
BEIJING UNIV OF AGRI
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AI Technical Summary

Problems solved by technology

The inventor also used the Percoll method to do a similar preliminary test in the breeding rooster in the early stage, and the results showed that the Leydig cells of the testes of the breeding cock were mainly concentrated in the 30% to 60% Percoll layer, but a pair of testis of a single breeding cock The amount of obtained cells in the Percoll layer is very small, and the activity rate and purity are very low

Method used

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  • Method for preparing poultry testicular interstitial cells
  • Method for preparing poultry testicular interstitial cells
  • Method for preparing poultry testicular interstitial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Isolation and culture of Leydig cells

[0045] 1. Procurement of testicular tissue

[0046] 1. Select healthy and disease-free breeding roosters with good mental state as test subjects.

[0047] 2. Bloodletting the jugular vein of the rooster to death, immersing in water containing brogeramine for 10-15 minutes, immediately taking out both testes after dissection (to maintain the integrity of the testicular tissue), and cutting off the excess tissue around the testis.

[0048] 3. Immediately after taking out both testes, place them in pre-cooled PBS buffer (pH7.2-7.4) containing 2% double antibodies (penicillin and streptomycin), and wash them three times with PBS. Afterwards, the isolated testicular tissue was placed in a small sterile beaker and quickly transferred to the intercellular ultra-clean bench.

[0049] attached dissection process : Put the breeding rooster on the operating table, lift the skin connecting the end of the sternum and the abdomen...

Embodiment 2

[0065] Example 2. Identification of cell viability by trypan blue staining

[0066] Preparation of 0.4% trypan blue dye solution: Accurately weigh 0.4g trypan blue powder, dissolve in 100mL PBS, shake and mix well, filter and package with 0.22μm filter membrane, store at 4°C for later use.

[0067] The Leydig cell suspension was stained with 0.4% trypan blue solution and observed under a light microscope. A total of 200 cells were counted, among which dead cells were stained blue and living cells were not stained. Repeat the count 3 times. According to the calculation results, if the cell viability is greater than 90%, it can be used for subsequent experiments.

[0068] Calculated as follows:

[0069] Cell viability = number of viable cells / (number of viable cells + number of dead cells) × 100%

[0070] The cell viability results are listed in Table 1 below.

Embodiment 33

[0071] Example 3.3 β-HSD staining method to identify the purity of Leydig cells

[0072] Preparation of 3β-HSD staining solution: mixed solution A: 0.6 mg dehydroepiandrosterone (DHEA) and 1 mg nitro blue tetrazolium (NBT) were dissolved in 0.6 mL methanol; mixed solution B: 10 mg coenzyme Ⅰ (NAD+) Dissolve in 9.5ml DPBS, mix liquid A and liquid B at a ratio of 0.6:9.5 (preparation for immediate use).

[0073] Leydig cells are specifically stained blue because only Leydig cells contain 3β-hydroxysteroid dehydrogenase (3β-HSD). In the present invention, the adherent cells of the third generation Leydig cells are selected for staining test for observation. First wash the cells carefully with DPBS, add 1.5-2mL 3β-HSD staining solution to the culture dish, put it in the incubator and continue to incubate for 5 hours, select multiple fields of view under the microscope to observe and count all the cells and the cells stained blue , to calculate the cell purity of Leydig cells.

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Abstract

The invention belongs to the field of cell separation and purification and more specifically relates to a method for preparing poultry testicular interstitial cells. The method comprises the followingsteps: digesting a testicular tissue block by utilizing collagenase to obtain a cell suspension solution; centrifuging the cell suspension solution obtained by step a) by utilizing a differential centrifugation method and separating to obtain the testicular interstitial cells; culturing the testicular interstitial cells obtained by step b) by utilizing a differential attachment method, so as to enrich the testicular interstitial cells. According to the method for preparing the poultry testicular interstitial cells, provided by the invention, the testicular interstitial cells with great cell amount and high purity can be obtained; an operation process is simple and standard, damages to the cells are relatively small and cell pollution is effectively reduced.

Description

technical field [0001] The invention belongs to the field of cell separation and purification. More specifically, the present invention relates to a method for preparing avian Leydig cells. Background technique [0002] Leydig cells (Leydig cells, LC) are distributed in the space of the seminiferous tubules of the testis and are the main place for the synthesis and secretion of androgen. Testosterone secreted by Leydig cells accounts for about 96% of the total testosterone in animals. Because testosterone can play many important physiological functions after binding to target organ receptors, Leydig cells are the most important cells that secrete testosterone, and their isolation and culture technology has attracted more and more attention. Therefore, establishing a stable, efficient and convenient method for the isolation, culture and purification of primary cells of Leydig cells is of great significance for in-depth research on the related functions of Leydig cells. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0683C12N2509/00
Inventor 齐晓龙郭勇陈余尚明玉陈晨盛熙晖
Owner BEIJING UNIV OF AGRI
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